The longterm objective of this proposal is to develop gene therapy approaches for the treatment of inborn errors of metabolism (IEM) that involve the transfer of plasmid DNA (pDNA) directly into muscle or liver. Plasmid DNA has been delivered directly into muscle as naked DNA or into liver as polylysine complexes. At least a ten-fold increase in the efficiency of gene expression into muscle or liver in vivo is required to treat IEM. The proposed work will focus on the movement of pDNA from the cytoplasm into nucleus, a transport step that our preliminary data indicates is rate limiting for efficient plasmid expression. The nuclear transport of pDNA occurred through the nuclear pore by a facilitative process not common to other large karyophilic macromolecules. The relatively rare entry of pDNA into the nucleus (in comparison to karyophilic proteins) was explained by pDNA's rapid and substantial cytoplasmic binding or restriction and pDNA's low rate of transport through the nuclear pore into the nucleus. The focus of this grant proposal is to study these two steps and use this knowledge to increase the amount of pDNA nuclear transport. Novel complexes of pDNA and molecules containing nuclear targeting signals (NTS) will be synthesized. The effect of these complexes on the nucleocytoplasmic transport of pDNA will be studied using state-of- the art fluorescent and electron microscopic techniques. The effect on reporter gene expression will be studied with some of the pDNA complexes. In a re-iterative fashion, the development of novel methods that attempt to increase pDNA nuclear transport will also aid the studies into the mechanism of pDNA nuclear transport and vice versa. Our proposed model systems for the study of pDNA nucleocytoplasmic transport has great significance and utility for other pDNA transfer methods.